核酸和蛋白样品制备

站在巨人的肩膀上,Fred Sanger两次获得诺贝尔化学奖,1958年因对胰岛素蛋白进行测序和1980年对DNA测序的贡献,其中都离不开illustra核酸及蛋白样品制备技术。

样品制备——即制备核酸或蛋白质样品用于后续分析——Amersham和illustra分子生物学产品在Sanger的两项研究中都扮演着关键的角色,自此成为了生命科学实验室高水平研究中的必备工具。

没有高质量的样品制备,就会导致后续实验结论不正确。比如,可能错过已降解的重要mRNA转录过程,而受损的酶会导致错误的动力学数据。

样品制备的方法通常基于样品的生理化学性质,比如在溶剂中的溶解性不同、是否沉淀、固相吸附、酶催化、高选择性亲和纯化,或者通过分子量大小和带电量不同进行分离。

运用一些特殊方法,从单独样品中分离出核酸和蛋白、甚至是两者同时提取分离。

样品制备在下游分析步骤中也是必要的,去除那些抑制或干扰下一步分析结果的颗粒物或杂相成分,可以最大程度减少错误、偏奇和误差。

    样品制备的目的是要得到一种最能满足您下游分析检测要求的样品,这意味着需获得更高纯度的样品、保留分子结构、维持生物活性,或确保感兴趣生物分子的相对含量。同时必须去除降解目标分子的组分,去除干扰影响下游分析的污染物,消除影响样品长期保存的因素。我们通常需要考虑如效率、时间、精力和成本因素。

    通常情况下,选择一个好品牌和专业支持,比如GE医疗生命科学的Whatman,Amersham,illustra,HiTrap等品牌,意味着离成功更近了一步。

    样品制备一般从最初提取的组织或细胞裂解液开始,要确保靶标生物分子降解最小化,必须创造一个保护性的环境,然后或通过抽提、研磨、除杂、抑制、脱盐、选择性吸附或浓缩的方式,进行样品进一步纯化。GE医疗生命科学提供蛋白和核酸制备的丰富产品和专业技术支持。

Much of GE Healthcare's filtration media are available in either flat format or in encapsulated formats, such as syringe filters, capsule filters, and filtration microplates.

  • Flat filters: Circles or sheet format. Circles would normally be used in an appropriate filter holder.
  • Prepleated filters (only for cellulose papers): Prefolded/pleated filter paper circles for use in conical filter funnels.
  • Syringe filters: Small, encapsulated, disposable filter units intended for filtration of small sample volumes using a syringe.
  • Syringeless filters/filter vials: Encapsulated, disposable filter units for small volume sample preparation not requiring the use of a syringe to drive the filtration step.
  • Inline filters: Encapsulated, disposable filter units intended for connection to flexible tubing. Units are available for filtration of liquids and for air/gas applications.
  • Capsule filters: Disposable filter units incorporating pleated filter media for larger volumes of liquid and air/gas applications.
  • Filtration microplates: Microplates with incorporated filter media for simultaneous filtration of multiple samples.

 

Filter characteristics and typical properties

The selection of a laboratory filter depends on the conditions and objectives of your experiment or analytical procedure. The three most important characteristics of any laboratory filter are:

  • Particle retention efficiency
  • Fluid flow rate through the filter
  • Loading capacity

According to your particular application, other important filter characteristics may also require examination, for example wet strength, chemical resistance, purity, and ash level. 

Typical properties of filter papers and glass fiber filters

Membrane materials

Chemical compatibility chart of membranes and housings


Filtration Methods for flat filters

  Gravity Vacuum Pressure
Cellulose Filter Papers Suitable Suitable Not generally used
Glass Microfiber Not suitable Suitable Suitable
Membranes Not suitable Suitable Suitable


Maximum practical volumes of cellulose filter paper circle sizes (quadrant folded)

Volume (ml) 15 20 35 75 135 300
Filter Diameter (mm) 90 110 125 150 185 240

 

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